Note: Descriptions are shown in the official language in which they were submitted.
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SEMAPHORIN MODULATION OF CELLULAR EFFLUX
This application claims priority to United States provisional patent
application serial number
60/152,914 filed September 8, 1999, now abandoned; United States provisional
patent application
serial number 60/156,257, filed September 27, 1999, now abandoned; and United
States provisional
patent application serial number 60/173,906 filed December 29, 1999, now
abandoned.
I. FIELD OF THE INVENTION
The present invention relates to compositions and methods useful in
manipulating cellular
efflux mechanisms resulting in multiple drug resistance (MDR). More
specifically, the present
invention relates to the use of semaphorin or semaphorin receptor
polypeptides, as well as
polynucleotides encoding these polypeptides, to modulate cellular efflux or
the MDR phenotype of
cells.
II. BACKGROUND OF THE INVENTION
In response to unavoidable continuous exposure to a frequently hostile
environment, cells have
developed a multitude of mechanisms to prevent entry or to accelerate exit of
noxious substances from
the infra-cellular space. This "cellular Darwinism" is accepted as a basic
tool of survival, but, once
applied by targeted cells to cytotoxic drugs, the phenomenon interferes with
the effectiveness of
chemotherapies for an array of diseases such as cancer and HIV. As the result
of a wide spectrum of
highly effective systems, drug resistance, whatever its source, is a prevalent
cause for
chemotherapeutic failure.
When cellular resistance to one drug results in resistance to a wide array of
chemical agents,
including those that are not related to the substance originally inducing the
resistance, the cell is
regarded as having developed multidrug resistance, or MDR. Thus, MDR is a
cellular phenomenon
characterized by resistance of the cell to cytotoxic substances. Generally,
MDR develops in response
to a specific cytotoxic substance, but then confers resistance to an array of
cytotoxic substances or
conditions. Cells that have developed MDR are considered MDR phenotypic cells,
and are further
described as those cells that have an increased ability, relative to non-MDR
cells, to survive in the
presence of cytotoxic substances or cytotoxic conditions. The increased
survival rates of MDR
phenotypic cells is characteristically due to an increased cellular capacity
to efflux or expel from the
cell substances that are either cytotoxic in themselves, or are present in the
cell in cytotoxic amounts,
thereby creating a cytotoxic condition for the cell. In an attempt to
understand and control MDR,
many investigators have studied the various mechanisms thought to drive it.
See Kellen, Alternative
Mechanisms of Multidru~ Resistance in Cancer, (1995). MDR phenotypes of cancer
or other cells
may arise as a result of MDR proteins, or MDR-like proteins, or various other
mechanisms involving
efflux pumps. Cellular efflux pumps involved in the development of MDR
phenotypic cells include
those that are able to efflux molecules of many different sizes and
compositions, as well as protons or
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chloride ions. For example, MDR protein pumps include the proteins MDR-1 and
MDR-2, which are
each considered to be a P-glycoprotein (P-gp), and the human multiple drug
resistance associated
protein designated "MRP" (see, Zaman, et al., 1994). These and other MDR
proteins are
transmembrane efflux pumps that, based on studies in the mouse, are believed
to be important in
S removing toxins from the cell.
Various assays that have been developed to allow the study of exchange of
molecules across
membranes are employed in the study of MDR proteins. For example, many
lipophilic, cationic dyes
have been described that allow one to follow changes in membrane potential, or
changes in
intracellular pH. One such dye, Rhodamine 123 (Rh123) was frequently used by
hematologists to
measure mitochondrial membrane potential, and has been described as a
substrate for MDR proteins.
Kim et.al., (1998). Consistent with the reported transport of protons,
expression of the MDR protein P-
gp has been associated with a significant elevation of intracellular pH
(Weisburg et al, 1999).
Further, MDR phenotypes are reported to arise in some cell types as a result
of alterations in
the acidification (pH) of intracellular organelles and compartments, such as
the trans-golgi network
1 S and the endocytic pathway (see, e.g., Altan, et al., Altan, N et al.,
Chen, Y et al., Schindler, et al.).
One mechanism for controlling the pH of intracellular compartments is by
cellular pumps that operate
to move protons, or negatively charged ions like chloride ions, across
membranes. Such cellular
pumps are implicated in certain diseases. For example, unregulated activity of
a chloride pump is
known to be at least partially responsible for the development of cystic
fibrosis resulting from a
genetic defect. Alternatively, growth factors are theorized to play a non-
efflux-related role in MDR.
For example, semaphorins have been postulated to function as growth factors,
and thereby exert an
effect on cells that may contribute to the development of drug resistance
(Yamada, et al.).
In light of the various relationships between cellular efflux pumps and MDR,
the ability to
control such efflux pumps would provide the ability to promote or suppress the
development of MDR
in cells. Accordingly, investigation into MDR mechanisms, and various methods
for controlling MDR
via control of cellular efflux mechanisms is ongoing.
III. SUMMARY OF THE INVENTION
The present invention teaches the use of semaphorin or semaphorin receptor
polypeptides to
modulate the activity of cellular efflux pumps. The present invention further
teaches that semaphorin
or semaphorin receptor polypeptides can be used to specifically activate or
inhibit cellular efflux
pumps and therefore may induce or inhibit the development of multiple drug
resistant cells. The
present invention further provides compositions and methods for the treatment
of neoplasms,
autoimmune or immuno-deficiency disorders such as HIV, and other cellular-
efflux-related disease
states.
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The present invention specifically contemplates that any semaphorin
polypeptide, or active
fragment of a semaphorin polypeptide, may be used in the disclosed
compositions and methods.
Exemplary semaphorins include, for example and without limitation: AHV Sema;
A39R; Sema I,
including G-sema I and D-sema-I; Sema II; Sema III; Sema IV; DC Sema; CD100; Z
SMF-7; Sema A;
Sema B; Sema C; Sema D; Sema E; Sema H; Sema L; Sema W and Sema Y.
Additionally, useful
fragments of any semaphorin, such as the sema domain or the active domain may
also be used
according to the present invention. For additional semaphorins that can be
used in the presently
disclosed compositions and methods, see Bamberg, et.al. Cell, 97:551 and
United States Patent No.
5,935,865 to Goodman et al. In alternative embodiments of the present
invention, such as in "gene
therapeutics," nucleic acid sequences encoding any of these semaphorins or
their fragments can be
used.
Similarly, preferred semaphorin receptor polypeptides for use in the presently
disclosed
compositions and methods include those semaphorin receptors known as plexins,
as well as their
complements, variants and useful fragments such as soluble portions of the
receptors, fragments
including the sema domain of the plexins, and fragments including the active
sites of the plexins. A
particularly preferred plexin for use according to the present invention is
the Viral-Encoded
Semaphorin Receptor ("VESPR"), as well as complements, variants, and soluble
fragments thereof.
Particularly preferred polypeptide sequences include the polypeptide sequence
of SEQ ID N0:2.
Additionally, useful soluble forms of the VESPR polypeptide include those
segments of the
polypeptide comprising a portion of the extracellular domain of the receptor.
An example of a soluble
VESPR polypeptide includes amino acids 1-944 of SEQ ID N0:2. In addition,
truncated soluble
VESPR proteins comprising less that the entire extracellular domain are
included in the invention, e.g.,
amino acids 35-944. Also encompassed within the present invention are the
nucleic acid sequences
encoding such useful VESPR polypeptides and polypeptide fragments.
Particularly preferred nucleic
acid sequences include the polynucleotide sequence of SEQ 1D NO:1; and those
segments of SEQ ID
NO:1 that encode the soluble fragments of VESPR outlined above. The VESPR, its
useful fragments,
complements, variants, and combinations, such as fusion proteins as well as
the nucleic acid sequences
encoding these polypeptides are described in co-pending application SN
08/958,598 (specifically
incorporated herein by reference, in its entirety). In embodiments of the
present invention employing
nucleic acid sequences, such as in "gene therapeutics," nucleic acid sequences
encoding any of these
semaphorin receptor polypeptides or their fragments can be used.
In a preferred embodiment, the present invention provides a pharmaceutical
composition for
the treatment of MDR phenotypic cells. This composition comprises an amount of
a semaphorin or a
semaphorin receptor polypeptide such that administration of the composition is
effective to modulate
the MDR phenotype of the target cells. Alternatively, in another aspect of the
invention, the
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composition further includes an amount of an expression vector including a
nucleic acid sequence
encoding a semaphorin, a semaphorin receptor, or a useful fragment of a
semaphorin or semaphorin
receptor, such that administration of the composition is effective to modulate
the MDR phenotype of
the target cell. This modulation may be to either promote or inhibit the
development of multiple drug
S resistant cells.
An alternative embodiment of the present invention provides another
pharmaceutical
composition for the treatment of MDR phenotypic cells. In this aspect, the
presently disclosed
composition includes an amount of an agonist or antagonist for a semaphorin or
a semaphorin
receptor, such that administration of the composition is effective to promote
or inhibit the development
of MDR phenotype. Exemplary agonists or antagonists for semaphorins or
semaphorin receptors
include antibodies, such as, for example, either polyclonal or monoclonal
antibodies, antigens and
small molecules.
For example, a composition of the present invention can use a semaphorin
antagonist, in the
form of a soluble semaphorin receptor for example, to inhibit induction or
activation of cellular efflux
pumps. Use of such a composition allows one to decrease the ability of a cell
to expel agents crossing
the cell membrane, such as cytotoxic therapeutic agents. Alternatively or
additionally, a composition
of the present invention can include an antibody to a semaphorin receptor such
as VESPR, which can
function as either an antagonist or an agonist, or a small molecule agonist of
a semaphorin receptor
such as VESPR can be used.
In another embodiment, the present invention provides a pharmaceutical
composition, for the
treatment of cellular efflux-related disease states. In this aspect, the
composition includes an amount
of a semaphorin or semaphorin receptor such that administration of the
composition is effective to
modulate cellular efflux. Alternatively, in this aspect of the invention, the
composition includes an
amount of an expression vector including a nucleic acid sequence encoding a
semaphorin, a
semaphorin receptor, or encoding a useful fragment of a semaphorin or
semaphorin receptor, such that
administration of the composition is effective to modulate cellular efflux of
the target cells. The active
polypeptide or nucleic acid sequences of the composition used in this aspect
of the invention may
function to activate or up-regulate, or to inhibit or down-regulate, cellular
efflux.
In an alternative embodiment, the present invention provides another
composition for the
treatment of cellular efflux-related disease states. In this embodiment, the
disclosed composition
includes an amount of an agonist or antagonist of a semaphorin or semaphorin
receptor, such that
administration of the composition is effective in activating or inhibiting
cellular efflux in the target
cell. Exemplary agonists or antagonists for semaphorins or semaphorin
receptors include antibodies,
such as, for example, either polyclonal or monoclonal antibodies; antigens and
small molecules.
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In another aspect, the present invention provides a method of modulating
cellular efflux by
administering to a cell an effective amount of a composition including a
semaphorin or semaphorin
receptor polypeptide such that cellular efflux is activated or inhibited.
Alternatively, the present
invention provides a method of modulating cellular efflux comprising
administering to a cell, via an
appropriate vector, an effective amount of a polynucleotide encoding a
semaphorin, a semaphorin
receptor, or a useful fragment of a semaphorin or semaphorin receptor, such
that cellular efflux is
activated or inhibited. Additionally, the presently disclosed methods of
modulating cellular efflux,
may comprise administering to a cell an effective amount of an agonist or
antagonist of a semaphorin
or semaphorin receptor such that cellular efflux is activated or inhibited.
Exemplary useful agonists or
antagonists include antibodies such as, for example, monoclonal or polyclonal
antibodies, an antigen,
or a small molecule. In a particularly preferred embodiment, the antibody used
is an antibody to
VESPR.
Pharmaceutical compositions and methods of the presently disclosed invention
may be useful
in the treatment of cellular efflux-related disease states such as multiple
drug resistance; cancers, or
other neoplastic diseases such as tumors, leukemia, lymphoma or other
localized or metastatic
conditions characterized by an abnormal proliferation of cells, generally due
to cells continuing to
replicate after the stimuli that initiated growth has ceased; cystic fibrosis
arising from the treatment of
a cell or group of cells with cytotoxic agents; auto-immune disorders; or
acquired or genetically-based
immunodeficiency disorders such as that resulting from the human
immunodeficiency virus (HIV).
Formulation of any of the presently disclosed compositions for administration
according to the
disclosed methods can be done in any manner known to those of skill in the
art. Such formulations
will vary according to variables such as, for example, the needs of the
formulator, the intended route of
administration, the targeted disease or tissue, and the subject being treated.
Specifically, unit doses
may be formulated in multi-dose containers including additives such as a
carrier, other excipients, and
a preservative component.
The disclosed compositions may be formulated in a variety of concentrations in
various vial
sizes for various administration dosages. The presently disclosed compositions
may also be in
virtually any form including an aqueous solution, a suspension, a lyophilized
form that may be
reconstituted when appropriate, a gel, an aerosol, or any other form or state
convenient for
administration to treat the described disorders. The compositions as described
herein may be
formulated so that they are contained in a vial, bottle, tube, syringe,
inhaler, transdermal patch, capsule
or other container for single or multiple administrations.
In alternative embodiments, the presently disclosed compositions are
formulated with or
administered in conjunction with additional active agents such as
chemotherapeutic agents, immune
suppressants or radiation therapy. For example, agents that may be useful to
co-formulate or
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administer in conjunction with the disclosed compositions include virtually
any chemotherapeutic or
sensitizing agent such as cyclosporin, FK506, taxotere, doxorubicin, cis-
platin, tamoxifen, i-
phosphamide, or methotrexate, or variants of any of these compounds.
Alternatively or additionally,
the presently disclosed compositions may be further co-administered with an
immune suppressant,
such as a cytokine, IL-4, IL-12, , GM-CSF, G-CSF, M-CSF, a-interferon, ~i-
interferon, or Y-
interferon. The additional agents may be co-administered simultaneously or
sequentially relative to
the disclosed compositions and methods.
In another aspect, the present invention provides various assays and screening
methods to
identify substances that may be used to influence the MDR phenotype of a cell.
For example, the
present invention provides a method of detecting the ability of a test
compound to affect the MDR
phenotype of a cell, in which the following steps are used: (1) contacting a
first cell with a test
compound and a semaphorin or a semaphorin receptor, in the presence of a
cytotoxic agent; (2)
measuring the rate of death of the first cell; (3) observing the rate of death
of a control cell in the
absence of the test compound; and (4) comparing the rate of death of the first
cell to the rate of death
1 S of the control cell. Upon comparison, a difference in the rate of cell
death of the first cell relative to
the control cell indicates that the test compound is an effector of MDR
phenotype. In this manner, the
effector can be identified as a substance that either promotes development of
MDR phenotype or
inhibits development of MDR phenotype. The affector can then be used
therapeutically.
Alternatively, the test compound may itself be a semaphorin or semaphorin
receptor or fragment or
antagonist or agonist thereof.
This method can be performed with a cytotoxic or sensitizing agent such as,
for example,
tamoxifen, cisplatin, doxorubicin, radiation, methotrexate, cyclosporin,
taxotere, FK506, or i-
phosphamide. Further, as with all compositions and methods of the present
invention, the semaphorin
or semaphorin receptor used in this method can be any known semaphorin or
receptor polypeptide or
useful fragment thereof, such as a fragment comprising the sema domain or the
active domain of a
semaphorin or semaphorin receptor. Additionally or alternatively, the
presently disclosed method can
be performed with any known semaphorin or semaphorin receptor, or fragment
thereof being the test
compound, or with an antibody to VESPR as the test compound.
In another aspect, the present invention provides a method of detecting the
ability of a test
compound to effect the MDR phenotype of a cell by modulating cellular efflux
in the cell. Such a
method would involve, for example, the following steps: (1) contacting a first
cell with a test
compound and a semaphorin or semaphorin receptor, in the presence of a dye;
(2) measuring the net
rate of influx of dye into the first cell; (3) observing the net rate of
influx of dye into a control cell, in
the absence of test compound comprising a semaphorin or semaphorin receptor,
under otherwise
identical conditions; and (4) comparing the net rate influx of dye into the
first cell to the net rate of
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influx of dye into the control cell. Upon comparison, a difference in the net
rate of influx of dye into
the first cell relative to the control cell indicates that the test compound
is an effector of cellular efflux.
In this manner, the effector can be identified as a substance that either
promotes cellular efflux or
inhibits cellular efflux and then can be used therapeutically. Alternatively,
the test compound may
S itself be a semaphorin or semaphorin receptor or fragment or antagonist or
agonist thereof.
Any dye may be used in the assays of the present invention. The dyes useful in
such methods
may be characterized by, for example, one or more of the following properties:
lipophilic, cationic,
fluorescent, and radioactive. Alternatively or additionally, the dye used in
such methods can be a slow
dye, a fast dye, acridine orange, BODIPY ceramide, SNARF-dextran, FITC-
transferrin or BODIPY-
transferrin.
As with all compositions and methods of the present invention, the semaphorin
or semaphorin
receptor used in this method can be any known semaphorin or semaphorin
receptor polypeptide or
useful fragment thereof, such as a fragment comprising the sema domain or the
active domain of a
semaphorin or semaphorin receptor. Additionally or alternatively, the
presently disclosed method can
be performed with any known semaphorin or semaphorin receptor, or fragment
thereof being the test
compound, or with an antibody to VESPR as the test compound.
In yet another aspect, the present invention provides pharmaceutical
compositions and
methods for the regulation of cellular-efflux, or MDR phenotype, by using the
agent identified by the
assays described herein. In this aspect of the invention, the modulating agent
is effective to either
inhibit or activate cellular efflux or development of drug resistance in a
target cell.
IV. DETAILED DESCRIPTION OF THE INVENTION
Contrary to the results of Yamada et al., who postulate that semaphorins
function analogously
to growth factors and may be involved in non-MDR drug resistance, the present
invention teaches that
semaphorins and semaphorin receptors can be used to influence the function of
cellular efflux pumps
in a variety of ways, including activation, inhibition, and promotion of
stasis of the pumps and can be
used to regulate MDR. The invention also teaches that, depending upon the
specific
semaphorin/receptor interaction, this influence can be inhibitory, and the
capacity of a cell to eliminate
cellular contents can be reduced, or the influence can be to promote cellular
efflux and thereby
facilitate expulsion of cellular contents. Accordingly, depending upon the
effect, the disclosed
semaphorin and semaphorin receptor compositions and methods are also useful:
(1) to increase
vulnerability or sensitivity of a cell to cytotoxic agents and thereby promote
drug-induced cell death;
(2) in identification or design of semaphorin or semaphorin receptor
antagonists or agonists that might
increase the sensitivity of a cell to a cytotoxic agent; (3) to promote
cellular resistance to cytotoxic
agents; or (4) in identification of semaphorin or semaphorin receptor agonists
or antagonists that can
be administered to cells to promote their resistance to various cytotoxic
substances.
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A. SEMAPHORIN AND SEMAPHORIN RECEPTOR POLYPEPTIDES
The terms "semaphorin" and "semaphorin polypeptide" are used interchangeably
in the
present invention. Semaphorins include proteins of the Semaphorin family and
are either secreted or
membrane-bound. Semaphorins have a well-conserved extracellular semaphorin
(sema) domain.
Generally, the sema domain is approximately 500 residues, but viral
semaphorins themselves are only
approximately 440 to 441 amino acids in length. It has been hypothesized that
a 70 amino acid region
with the sema domain is the active domain for semaphorin influence on certain
cellular activities. See
Koppel, et al. (1997). However it is not clear that this same region is the
active site for all semaphorin
activity. Accordingly, the present invention specifically contemplates the use
of full-length
semaphorin polypeptides, variants of these, and useful fragments of semaphorin
polypeptides.
Specific semaphorins and semaphorin fragments that are useful according to the
present invention
include, for example, the following semaphorins: AHV Sema; A39R; Sema I,
including G-sema I and
D-sema-I; Sema II; Sema III; Sema IV; DC Sema; CD100; Z SMF-7; Sema A; Sema B;
Sema C;
Sema D; Sema E; Sema H; Sema L; Sema W and Sema Y. Additionally, useful
fragments of any
1 S semaphorin, such as the sema domain or the active domain may also be used
according to the present
invention. For additional semaphorins that can be used in the presently
disclosed compositions and
methods, see Bamberg, et.al. Cell, 97:551 and United States Patent No.
5,935,865 to Goodman et al.
Nucleic acid sequences encoding the semaphorins or semaphorin fragments of the
present invention,
are also specifically contemplated to be useful in the disclosed compositions
and methods.
"Semaphorin receptors" or "semaphorin receptor polypeptides" of the present
invention are
members of the Plexin family of semaphorin receptors. Plexins are membrane-
bound polypeptides.
Plexins contain a "sema" domain that is related to the sema domain of
semaphorins themselves, part of
which constitutes a series of two or three cystein repeat sequences in the
extracellular domain of
plexins. Plexins are distinct from semaphorins, however, in a variety of
respects. For example, in
their intracellular domain, plexins are strongly homologous throughout the
family of plexins, and
contain well-conserved amino acid motifs that are not found in semaphorins.
Semaphorin receptors of the present invention are those plexin polypeptide
sequences that can
interact with a semaphorin or a semaphorin fragment, to influence cellular
efflux or development of
MDR phenotype in a cell Exemplary semaphorin receptor polypeptides include
full-length plexin
receptor polypeptides as well as homologues or fragments, such as the soluble
extra cellular domain or
the sema domain of such plexin receptor polypeptides. Preferred semaphorin
receptor polypeptides
include the Viral Encoded Semaphorin Receptor (VESPR) or fragments thereof. As
used herein, the
term VESPR or VESPR polypeptide refers to any polypeptide functioning as a
receptor for viral
semaphorins, for human homologues to viral semaphorins, or for human
semaphorins.
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Additionally, useful soluble forms of the VESPR polypeptide include those
segments of the
polypeptide comprising a portion of the extracellular domain of the receptor.
An example of a soluble
VESPR polypeptide includes amino acids 1-944 of SEQ ID N0:2. In addition,
truncated soluble
VESPR proteins comprising less that the entire extracellular domain are
included in the invention, e.g.,
S amino acids 35-944. Also encompassed within the present invention are the
nucleic acid sequences
encoding such useful VESPR polypeptides and polypeptide fragments. An
exemplary Plexin receptor
is the Viral Encoded Semaphorin Protein Receptor "VESPR," (described in
copending patent
application serial number 08/958,598). Specifically the amino acid sequence of
SEQ ID N0:2 is
useful as a semaphorin receptor polypeptide in the presently disclosed
compositions and methods, as
are the homologues and variants of polypeptides of SEQ ID N0:2. Nucleic acid
sequences encoding
the semaphorin receptors or receptor fragments are also within the scope of
the presently disclosed
compositions and methods. Particularly preferred nucleic acid sequences
include the polynucleotide
sequence of SEQ ID NO:I; and those segments of SEQ ID NO:1 that encode the
soluble fragments of
VESPR outlined above.
The semaphorin or semaphorin receptor polypeptides of the invention may be
membrane
bound or they may be secreted and thus soluble. Soluble polypeptides are
capable of being secreted
from the cells in which they are expressed. In general, soluble polypeptides
may be identified (and
distinguished from non-soluble membrane-bound counterparts) by separating
intact cells which
express the desired polypeptide from the culture medium, e.g., by
centrifugation, and assaying the
medium (supernatant) for the presence of the desired polypeptide. The presence
of polypeptide in the
medium indicates that the polypeptide was secreted from the cells and thus is
a soluble form of the
protein.
In one embodiment, the soluble polypeptides and fragments thereof comprise all
or part of the
extracellular domain, but lack the transmembrane region that would cause
retention of the polypeptide
on a cell membrane. A soluble polypeptide may include the cytoplasmic domain,
or a portion thereof,
as long as the polypeptide is secreted from the cell in which it is produced.
In general, the use of soluble forms is advantageous for certain applications.
Purification of
the polypeptides from recombinant host cells is facilitated, since the soluble
polypeptides are secreted
from the cells. Further, soluble polypeptides are generally more suitable for
intravenous
administration.
The invention also provides polypeptides and fragments of the extracellular
domain that retain
a desired biological activity. Particular embodiments are directed to
polypeptide fragments that retain
the ability to interact with the semaphorin receptor or ligand to influence
cellular efflux or the MDR
phenotype of a cell. Such a fragment may be a soluble polypeptide, as
described above. In another
embodiment, the polypeptides and fragments advantageously include regions that
are conserved in the
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semaphorin family, in the case of semaphorins; or regions that are conserved
in the plexin family in
the case of the semaphorin receptors; or include the sema domain of either.
Also provided herein are polypeptide fragments comprising at least 20, or at
least 30,
contiguous amino acids of the sequence of SEQ ID N0:2. Fragments derived from
the cytoplasmic
domain fmd use in studies of signal transduction, and in regulating cellular
processes associated with
transduction of biological signals. Polypeptide fragments also may be employed
as immunogens, in
generating antibodies.
Naturally occurring variants as well as derived variants of the polypeptides
and fragments are
provided herein. Variants may exhibit amino acid sequences that are at least
80% identical. Also
contemplated are embodiments in which a polypeptide or fragment comprises an
amino acid sequence
that is at least 90% identical, at least 95% identical, at least 98%
identical, at least 99% identical, or at
least 99.9% identical to the preferred polypeptide or fragment thereof.
Percent identity can be
determined by visual inspection and mathematical calculation. Alternatively,
the percent identity of
two protein sequences can be determined by comparing sequence information
using the GAP computer
program, based on the algorithm of Needleman and Wunsch (J. Mol. Bio. 48:443,
1970) and available
from the University of Wisconsin Genetics Computer Group (UWGCG). The
preferred default
parameters for the GAP program include: (1) a scoring matrix, blosum62, as
described by Henikoff
and Henikoff (Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) a gap weight of
12; (3) a gap length
weight of 4; and (4) no penalty for end gaps. Other programs used by one
skilled in the art of
sequence comparison may also be used.
The variants of the invention include, for example, those that result from
alternate mRNA
splicing events or from proteolytic cleavage. Alternate splicing of mRNA may,
for example, yield a
truncated but biologically active protein, such as a naturally occurring
soluble form of the protein.
Variations attributable to proteolysis include, for example, differences in
the N- or C-termini upon
expression in different types of host cells, due to proteolytic removal of one
or more terminal amino
acids from the protein (generally from 1-5 terminal amino acids). Proteins in
which differences in
amino acid sequence are attributable to genetic polymorphism (allelic
variation among individuals
producing the protein) are also contemplated herein.
Additional variants within the scope of the invention include polypeptides
that may be
modified to create derivatives thereof by forming covalent or aggregative
conjugates with other
chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups
and the like. Covalent
derivatives may be prepared by linking the chemical moieties to functional
groups on amino acid side
chains or at the N-terminus or C-terminus of a polypeptide. Conjugates
comprising diagnostic
(detectable) or therapeutic agents attached thereto are contemplated herein,
as discussed in more detail
below.
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Other derivatives include covalent or aggregative conjugates of the
polypeptides with other
proteins or polypeptides, such as by synthesis in recombinant culture as N-
terminal or C-terminal
fusions. Examples of fusion proteins are discussed below in connection with
oligomers. Further,
fusion proteins can comprise peptides added to facilitate purification and
identification. Such peptides
S include, for example, poly-His or the antigenic identification peptides
described in U.S. Patent No.
5,011,912 and in Hopp et al., BiolTechnology 6:1204, 1988. One such peptide is
the FLAG~ peptide,
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, which is highly antigenic and provides an
epitope reversibly
bound by a specific monoclonal antibody, enabling rapid assay and facile
purification of expressed
recombinant protein. A murine hybridoma designated 4E11 produces a monoclonal
antibody that
binds the FLAG~ peptide in the presence of certain divalent metal canons, as
described in U.S. Patent
5,011,912, hereby incorporated by reference. The 4E11 hybridoma cell line has
been deposited with
the American Type Culture Collection under accession no. HB 9259. Monoclonal
antibodies that bind
the FLAG~ peptide are available from Eastman Kodak Co., Scientific Imaging
Systems Division, New
Haven, Connecticut.
Among the variant polypeptides provided herein are variants of native
polypeptides that retain
the native biological activity or the substantial equivalent thereof. One
example is a variant that binds
with essentially the same binding affinity as does the native form. Binding
affinity can be measured
by conventional procedures, e.g., as described in U.S. Patent No. 5,512,457
and as set forth below.
Variants include polypeptides that are substantially homologous to the native
form, but which
have an amino acid sequence different from that of the native form because of
one or more deletions,
insertions or substitutions. Particular embodiments include, but are not
limited to, polypeptides that
comprise from one to ten deletions, insertions or substitutions of amino acid
residues, when compared
to a native sequence.
A given amino acid may be replaced, for example, by a residue having similar
physiochemical
characteristics. Examples of such conservative substitutions include
substitution of one aliphatic
residue for another, such as Ile, Val, Leu, or Ala for one.another;
substitutions of one polar residue for
another, such as between Lys and Arg, Glu and Asp, or Gln and Asn; or
substitutions of one aromatic
residue for another, such as Phe, Trp, or Tyr for one another. Other
conservative substitutions, e.g.,
involving substitutions of entire regions having similar hydrophobicity
characteristics, are well known.
Similarly, the DNAs of the invention include variants that differ from a
native DNA sequence
because of one or more deletions, insertions or substitutions, but that encode
a biologically active
polypeptide.
The invention further includes polypeptides of the invention with or without
associated native-
pattern glycosylation. Polypeptides expressed in yeast or mammalian expression
systems (e.g., COS-1
or COS-7 cells) can be similar to or significantly different from a native
polypeptide in molecular
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weight and glycosylation pattern, depending upon the choice of expression
system. Expression of
polypeptides of the invention in bacterial expression systems, such as E.
coli, provides non-
glycosylated molecules. Further, a given preparation may include multiple
differentially glycosylated
species of the protein. Glycosyl groups can be removed through conventional
methods, in particular
those utilizing glycopeptidase. In general, glycosylated polypeptides of the
invention can be incubated
with a molar excess of glycopeptidase (Boehringer Mannheim).
Correspondingly, similar DNA constructs that encode various additions or
substitutions of
amino acid residues or sequences, or deletions of terminal or internal
residues or sequences are
encompassed by the invention. For example, N-glycosylation sites in the
polypeptide extracellular
domain can be modified to preclude glycosylation, allowing expression of a
reduced carbohydrate
analog in mammalian and yeast expression systems. N-glycosylation sites in
eukaryotic polypeptides
are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino
acid except Pro and Y is
Ser or T'hr. Appropriate substitutions, additions, or deletions to the
nucleotide sequence encoding
these triplets will result in prevention of attachment of carbohydrate
residues at the Asn side chain.
Alteration of a single nucleotide, chosen so that Asn is replaced by a
different amino acid, for
example, is sufficient to inactivate an N-glycosylation site. Alternatively,
the Ser or Thr can by
replaced with another amino acid, such as Ala. Known procedures for
inactivating N-glycosylation
sites in proteins include those described in U.S. Patent 5,071,972 and EP
276,846, hereby incorporated
by reference.
In another example of variants, sequences encoding Cys residues that are not
essential for
biological activity can be altered to cause the Cys residues to be deleted or
replaced with other amino
acids, preventing formation of incorrect intramolecular disulfide bridges upon
folding or renaturation.
Other variants are prepared by modification of adjacent dibasic amino acid
residues, to
enhance expression in yeast systems in which KEX2 protease activity is
present. EP 212,914 discloses
the use of site-specific mutagenesis to inactivate KEX2 protease processing
sites in a protein. KEX2
protease processing sites are inactivated by deleting, adding or substituting
residues to alter Arg-Arg,
Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic
residues. Lys-Lys
pairings are considerably less susceptible to KEX2 cleavage, and conversion of
Arg-Lys or Lys-Arg to
Lys-Lys represents a conservative and preferred approach to inactivating KEX2
sites.
Encompassed by the invention are oligomers or fusion proteins that contain
semaphorin or
semaphorin receptor polypeptides. Such oligomers may be in the form of
covalently-linked or non-
covalently-linked multimers, including dimers, trimers, or higher oligomers.
As noted above,
preferred polypeptides are soluble and thus these oligomers may comprise
soluble polypeptides. In
one aspect of the invention, the oligomers maintain the binding ability of the
polypeptide components
and provide therefor, bivalent, trivalent, etc., binding sites.
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One embodiment of the invention is directed to oligomers comprising multiple
polypeptides
joined via covalent or non-covalent interactions between peptide moieties
fused to the polypeptides.
Such peptides may be peptide linkers (spacers), or peptides that have the
property of promoting
oligomerization. Leucine zippers and certain polypeptides derived from
antibodies are among the
peptides that can promote oligomerization of the polypeptides attached
thereto, as described in more
detail below.
As one alternative, an oligomer is prepared using polypeptides derived from
immunoglobulins.
Preparation of fusion proteins comprising certain heterologous polypeptides
fused to various portions
of antibody-derived polypeptides (including the Fc domain) has been described,
e.g., by Ashkenazi et
al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and
Hollenbaugh and Aruffo
("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in
Immunology, Suppl. 4,
pages 10.19.1 - 10.19.11, 1992).
One embodiment of the present invention is directed to a dimer comprising two
fusion
proteins created by fusing a polypeptide of the invention to an Fc polypeptide
derived from an
antibody. A gene fusion encoding the polypeptide/Fc fusion protein is inserted
into an appropriate
expression vector. Polypeptide/Fc fusion proteins are expressed in host cells
transformed with the
recombinant expression vector, and allowed to assemble much like antibody
molecules, whereupon
interchain disulfide bonds form between the Fc moieties to yield divalent
molecules.
The term "Fc polypeptide" as used herein includes native and mutein forms of
polypeptides
made up of the Fc region of an antibody comprising any or all of the CH
domains of the Fc region.
Truncated forms of such polypeptides containing the hinge region that promotes
dimerization are also
included. Preferred polypeptides comprise an Fc polypeptide derived from a
human IgGI antibody.
One suitable Fc polypeptide, described in PCT application WO 93/10151 (hereby
incorporated
by reference), is a single chain polypeptide extending from the N-terminal
hinge region to the native
C-terminus of the Fc region of a human IgGI antibody. Another useful Fc
polypeptide is the Fc
mutein described in U.S. Patent 5,457,035 and in Baum et al., (EMBO J. 13:3992-
4001, 1994)
incorporated herein by reference. The amino acid sequence of this mutein is
identical to that of the
native Fc sequence presented in WO 93/10151, except that amino acid 19 has
been changed from Leu
to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has
been changed from
Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.
The above-described fusion proteins comprising Fc moieties (and oligomers
formed
therefrom) offer the advantage of facile purification by affinity
chromatography over Protein A or
Protein G columns.
In other embodiments, the polypeptides of the invention may be substituted for
the variable
portion of an antibody heavy or light chain. If fusion proteins are made with
both heavy and light
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chains of an antibody, it is possible to form an oligomer with as many as four
semaphorin or
semaphorin receptor extracellular regions.
Alternatively, the oligomer is a fusion protein comprising multiple
polypeptides, with or
without peptide linkers (spacer peptides). Among the suitable peptide linkers
are those described in
U.S. Patents 4,751,180 and 4,935,233, which are hereby incorporated by
reference. A DNA sequence
encoding a desired peptide linker may be inserted between, and in the same
reading frame as, the DNA
sequences of the invention, using any suitable conventional technique. For
example, a chemically
synthesized oligonucleotide encoding the linker may be ligated between the
sequences. In particular
embodiments, a fusion protein comprises from two to four soluble semaphorin or
semaphorin receptor
polypeptides, separated by peptide linkers.
Another method for preparing the oligomers of the invention involves use of a
leucine zipper.
Leucine zipper domains are peptides that promote oligomerization of the
proteins in which they are
found. Leucine zippers were originally identified in several DNA-binding
proteins (Landschulz et al.,
Science 240:1759, 1988), and have since been found in a variety of different
proteins. Among the
known leucine zippers are naturally occurring peptides and derivatives thereof
that dimerize or
trimerize. Zipper domains (also referred to herein as an oligomerizing, or
oligomer-forming, domain)
and their use are well-known in the art.
B. ASSAYS
The influence of semaphorins on cellular efflux may be used to control the
development of
MDR phenotypes of a cell or group of cells. For example, semaphorin
polypeptides, or
polynucleotides encoding semaphorin polypeptides may be administered to a cell
or group of cells to
stimulate or inhibit cellular efflux, to either induce, enhance, suppress or
arrest the development of
multiple drug resistance in the target cells. Identification of semaphorin-
containing compositions that
may be used in this manner may be carried out via a variety of assays known to
those skilled in the art.
Included in such assays are those that evaluate the ability of a semaphorin
composition to influence
cell survival rates in the presence of cytotoxic agents. Such an assay would
involve, for example, the
determination of sensitivities of tumor cells or cell lines to anticancer
drugs in the presence and
absence of a semaphorin. In these assays, one would determine a rate of cell
death in the presence of
the cytotoxic agent (such as doxorubicin, etc.) and then determine if the rate
of cell death resulting
from that agent is altered in the presence of a semaphorin.
Alternatively, one might monitor MDR protein-like activity in a cell by
examining the ability
of primary cells, or cells overexpressing MDR proteins, to efflux dyes in the
presence and absence of a
semaphorin. These types of assays are routine, and employ what are referred to
as either "slow" or
"fast" cellular dyes, that is, dyes that are typically lipophilic, or
cationic. (see, e.g., Lelong, et al.,
1991). One example of use of these dyes involves loading the dye into a cell
at low temperatures, such
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as 4 degrees (or on ice), and then examining the stained cells by flow
cytometry. The cells will
fluoresce depending on how much dye they take up; and, if loaded in the
presence of an MDR efflux
pump inhibitor such as verapamil or cyclosporin, or a semaphorin of the
present invention, they may
fluoresce more brightly than cells loaded in the absence of an MDR inhibitor.
In this manner
S inhibitors of cellular efflux pumps can be identified. This assay may then
also be taken a step further
by transferring the cells loaded with dye to elevated temperature conditions,
such as 37 degrees
Centigrade, for a period of time, such as approximately three hours, at which
time the cells are again
examined on the flow cytometer, and compared to cells that were loaded with
dye and held at cooler
temperatures, such as the previously noted 4 degrees. At higher temperatures,
cellular efflux pumps,
including the MDR proteins, are quite active and can extrude the dye from the
cell at a rapid rate. The
ability of a semaphorin to influence this efflux can be measured by including
the semaphorin in the
assay during the efflux phase.
Yet another assay that may be used in the present invention involves examining
intracellular
pH, and the pH of intracellular compartments, in response to semaphorins.
These types of assays
again use fluorescent probes that target to the cytoplasm or to specific
organelles, and exhibit
fluorescence pattern changes as the pH changes. See, e.g. Altan, N et al. J.
Exp. Med. 187:1583,
Altan, N et al. PNAS 96:4432, Chen, Y et al. JBC 274:18364, Schindler, M. et
al. Biochemistry
35:2811.. Dyes that are useful in such assays include dyes such as acridine
orange (which targets
acidic compartments and whose fluorescent wavelength and intensity depends on
the pH of that
organelle), BODIPY-ceramide (which targets the trans-golgi network), SNARF-
dextrans of varying
molecular weights (allowing one to target cytosol or nucleus), and FITC-
transferrin or BODIPY-
transferrin (which targets endocytic vesicles). These dyes are used to stain
cells and then their
fluorescence intensity and/or pattern is measured on a confocal microscope.
Another embodiment of the present invention provides a method of detecting the
ability of the
test compound to influence the MDR phenotype of a cell. In this aspect of the
invention, the method
includes contacting a first cell with a test compound including a semaphorin
or semaphorin receptor in
the presence of a cytotoxic agent. The method then involves measuring the rate
of death of that first
cell. Then the rate of death of a controlled cell is observed, with the
control cell under similar
conditions but in the absence of a test compound comprising a semaphorin or
semaphorin receptor,
and in the presence of a cytotoxic agent, which is the same agent administered
to the first cell. The
death rate of the first cell is then compared to the death rate of the control
cell and the difference in the
rate of cell death between the first cell and the control cell is indicative
of an agent that influences
development of multiple drug resistance phenotype. This agent may be one that
functions to increase
multiple drug resistance in the cell or to decrease multiple drug resistance
in the cell.
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In addition to those semaphorins listed in Section III above, specific
semaphorins that may be
tested according to this embodiment of the invention include A39R, DCSema,
CD100, Sema III, Sema
E, or active fragments of these semaphorins. Specific exemplary useful
semaphorin receptors that may
be used in these assays include VESPR. Alternatively, an agonist or antagonist
to a semaphorin or
semaphorin receptor may used according to this aspect of the invention. In a
particularly preferred
embodiment, an antibody to VESPR is used. Examples of cytotoxic agents that
may be used in this
method of detection include doxorubicin, radiation, tamoxifen, or any other
compound known to have
a cytotoxic effect on a cell.
In another aspect, the present invention provides a method of detecting the
ability of a test
compound to influence the MDR phenotype of a cell by modulating the cellular
efflux of that cell. In
this aspect, one example of such a method includes: (1) contacting a first
cell with a test compound
including a semaphorin or semaphorin receptor in the presence of a dye; (2)
measuring the net rate of
influx of dye into this first cell; and (3) observing the net rate of influx
of dye into a control cell under
similar conditions, but in the absence of a test compound comprising a
semaphorin or semaphorin
receptor. In this embodiment, the net rate of influx of dye is the rate of
influx of dye relative to the
rate of efflux, as measured by the amount of dye detected in the cell. The
comparison will provide a
difference in the net rate of influx of the dye such that influx of the dye
into the first cell relative to the
control cell is indicative of an agent that can influence cellular efflux. The
test compound may
function to either activate or up-regulate, or inhibit or down-regulate
cellular efflux, either of which
function may be detected through this method.
In addition to the semaphorins listed in Section III above, specific
semaphorins that may be
tested according to this embodiment of the invention include A39R, DCSema,
CD100, Sema III, Sema
E, or active fragments of these semaphorins. Specific exemplary useful
semaphorin receptors that may
be used in these assays include VESPR. Alternatively, an agonist or antagonist
to a semaphorin or
semaphorin receptor may used according to this aspect of the invention. In a
particularly preferred
embodiment, an antibody to VESPR is used.
Virtually any dye may be used in this method. Exemplary dyes include those
which are
characterized by one or more of the following properties: lipophilic,
cationic, fluorescent, and
radioactive. Specific exemplary dyes include a slow dye; a fast dye; acridine
orange; various BODIPY
dyes including specific ones such as 4,4-difluoro-4-bora-3a,4a-diaza-s-
indacene-3,5-dipropionic acid,
BODIPY ceramide, and BODIPY-transferrin; seminaphthorhodafluors ("SNARF") -
dextran; and
Fluorescien isothiocyanate ("FITC")-transferrin.
C. COMPOUNDS AND METHODS FOR THE MODULATION OF CELLULAR EFFLUX
Described below are methods and compositions employing semaphorins, semaphorin
receptors, fragments of these, or the genes encoding them, for use in the
promotion or suppression of
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cellular efflux or for controlling development of MDR in a target cell or
group of cells. It is
specifically contemplated that such compositions and methods can be used to
treat a cell or group of
cells both in vivo and in vitro.
For example, such methods can comprise administering compounds which modulate
cellular
efflux, and thereby influence development of MDR phenotype or cellular efflux-
related disease states.
Administration of such compounds can be used to inhibit drug resistance
thereby sensitizing cells to
cytotoxic substances; to promote resistance to cytotoxic substances and
protect against cytotoxic
substances; or to the dysregulation of cellular efflux in cells that are
unable to otherwise regulate
themselves, such as those cells associated with diseases such as cystic
fibrosis.
In addition to methods utilizing semaphorin or semaphorin receptor-encoding
nucleic acid
sequences, it is also useful to modulate cellular efflux by using the
semaphorin or semaphorin receptor
polypeptide, or polypeptide fragments. Another means of modulating cellular
efflux or MDR
phenotypes according to the present invention involves the use of any of the
compounds identified
through the assays set forth in Section B above.
1 S When the actual nucleic acid sequences encoding the semaphorins;
semaphorin receptors; or
fragments of either that are disclosed in the present invention are delivered
according to the methods
described herein, it is advantageous to use a delivery mechanism so that the
sequences will be
incorporated into a cell for expression. Delivery systems that may
advantageously be employed in the
contemplated methods include the use of, for example, viral delivery systems
such as retroviral and
adenoviral vectors, as well as non-viral delivery systems. Such delivery
systems are well known by
those skilled in the art.
In one aspect of the invention, a retroviral delivery system may be employed.
The retroviruses
are a group of single-stranded RNA viruses characterized by an ability to
convert their RNA to
double-stranded DNA in infected cells by a process of reverse-transcription
(Coffin, 1990). The resulting
DNA then stably integrates into cellular chromosomes as a provirus and directs
synthesis of viral proteins.
The integration results in the retention of the viral gene sequences in the
recipient cell and its descendants.
The retroviral genome contains three genes - gag, pol, and env - that code for
capsid proteins, polymerise
enzyme, and envelope components, respectively. A sequence found upstream from
the gag gene, termed
y~, functions as a signal for packaging of the genome into virions. Two long
terminal repeat (LTR)
sequences are present at the 5' and 3' ends of the viral genome. These contain
strong promoter and
enhancer sequences and are also required for integration in the host cell
genome (Coffin, 1990).
1n order to construct a retroviral vector, a nucleic acid encoding a Crrb2 or
Crkl antisense
construct is inserted into the viral genome in the place of certain viral
sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging cell line
containing the gag, pol and env
genes but without the LTR and y~ components is constructed (Mann et al.,
1983). When a recombinant
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plasmid containing an inserted DNA, together with the retroviral LTR and yr
sequences, is introduced into
this cell line (by calcium phosphate precipitation for example), the y
sequence allows the RNA transcript
of the recombinant plasmid to be packaged into viral particles, which are then
secreted into the culture
media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The
media containing the
recombinant retroviruses is then collected, optionally concentrated, and used
for gene transfer. Retroviral
vectors are able to infect a broad variety of cell types. However, integration
and stable expression require
the division of host cells (Paskind et al., 1975).
Alternatively, an adenoviral delivery system may be employed. Human
adenoviruses are
double-stranded DNA tumor viruses with genome sizes of approximate 36 kB
(Tooze, 1981). As a model
system for eukaryotic gene expression, adenoviruses have been widely studied
and well characterized,
which makes them an attractive system for development of adenovirus as a gene
transfer system. This
group of viruses is easy to grow and manipulate, and they exhibit a broad host
range in vitro and in vivo.
In lytically infected cells, adenoviruses are capable of shutting off host
protein synthesis, directing cellular
machineries to synthesize large quantities of viral proteins, and producing
copious amounts of virus.
The El region of the genome includes ElA and E1B which encode proteins
responsible for
transcription regulation of the viral genome, as well as a few cellular genes.
E2 expression, including
E2A and E2B, allows synthesis of viral replicative functions, e.g. DNA-binding
protein, DNA
polymerase, and a terminal protein that primes replication. E3 gene products
prevent cytolysis by
cytotoxic T cells and tumor necrosis factor and appear to be important for
viral propagation. Functions
associated with the E4 proteins include DNA replication, late gene expression,
and host cell shutoff. The
late gene products include most of the virion capsid proteins, and these are
expressed only after most of
the processing of a single primary transcript from the major late promoter has
occurred. The major late
promoter (MLP) exhibits high efficiency during the late phase of the infection
(Stratford-Perricaudet and
Perricaudet, 1991 ).
As only a small portion of the viral genome appears to be required in cis
(Tooze, 1981),
adenovirus-derived vectors offer excellent potential for the substitution of
large DNA fragments when
used in connection with cell lines such as 293 cells. Ad5-transformed human
embryonic kidney cell lines
(Graham, et al., 1977) have been developed to provide the essential viral
proteins in traps.
Particular advantages of an adenovirus system for delivering foreign proteins
to a cell include (i)
the ability to substitute relatively large pieces of viral DNA by foreign DNA;
(ii) the structural stability of
recombinant adenoviruses; (iii) the safety of adenoviral administration to
humans; and (iv) lack of any
known association of adenoviral infection with cancer or malignancies; (v) the
ability to obtain high titers
of the recombinant virus; and (vi) the high infectivity of adenovirus.
Further advantages of adenovirus vectors over retroviruses include the higher
levels of gene
3 S expression. Additionally, adenovirus replication is independent of host
gene replication, unlike retroviral
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sequences. Because adenovirus transforming genes in the E1 region can be
readily deleted and still
provide efficient expression vectors, oncogenic risk from adenovirus vectors
is thought to be negligible
(Gnmhaus & Horwitz, 1992).
In general, adenovirus gene transfer systems are based upon recombinant,
engineered adenovirus
which is rendered replication-incompetent by deletion of a portion of its
genome, such as E1, and yet still
retains its competency for infection. Sequences encoding relatively large
foreign proteins can be
expressed when additional deletions are made in the adenovirus genome. For
example, adenoviruses
deleted in both E1 and E3 regions are capable of carrying up to 10 kB of
foreign DNA and can be grown
to high titers in 293 cells (Stratford-Perncaudet and Perricaudet, 1991).
Surprisingly persistent expression
of transgenes following adenoviral infection has also been reported.
Other viral vectors may be employed as expression constructs in the present
invention. Vectors
derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and
Sugden, 1986; Coupar et al.,
1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;
Hermonat and
Muzycska, 1984) and herpesviruses may be employed. They offer several
attractive features for various
mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986;
Coupar et al., 1988;
Norwich et al., 1990).
With the recent recognition of defective hepatitis B viruses, new insight was
gained into the
structure-function relationship of different viral sequences. In vitro studies
showed that the virus could
retain the ability for helper-dependent packaging and reverse transcription
despite the deletion of up to
80% of its genome (Norwich et al., 1990). This suggested that large portions
of the genome could be
replaced with foreign genetic material. The hepatotropism and persistence
(integration) were particularly
attractive properties for liver-directed gene transfer. Chang et al. recently
introduced the chloramphenicol
acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place
of the polymerase, surface,
and pre-surface coding sequences. It was cotransfected with wild-type virus
into an avian hepatoma cell
line. Culture media containing high titers of the recombinant virus were used
to infect primary duckling
hepatocytes. Stable CAT gene expression was detected for at least 24 days
after h-ansfection (Chang et
al., 1991).
In yet another aspect, non-viral vectors may be used according to the
presently disclosed methods.
Several non-viral methods for the transfer of expression vectors into cultured
mammalian cells also are
contemplated by the present invention. These include calcium phosphate
precipitation (Graham and Van
Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,
1985),
electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct
microinjection (Harland and Weintraub,
1985); DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and
lipofectamine-DNA
complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using
high velocity
microprojectiles (Yang et al., 1990), polycations (Boussif et al., 1995) and
receptor-mediated transfection
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(Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be
successfully adapted for in
vivo or ex vivo use.
In one embodiment of the invention, the expression construct may simply
consist of naked
recombinant vector. Transfer of the construct may be performed by any of the
methods mentioned above
S which physically or chemically permeabilize the cell membrane. For example,
Dubensky et al. ( 1984)
successfully injected polyomavirus DNA in the form of CaP04 precipitates into
liver and spleen of adult
and newborn mice demonstrating active viral replication and acute infection.
Benvenisty and Neshif
(1986) also demonstrated that direct intraperitoneal injection of CaP04
precipitated plasmids results in
expression of the transfected genes. It is envisioned that DNA encoding an
Grb2 or Crkl construct may
also be transferred in a similar manner in vivo.
Another embodiment of the invention for transferring a naked DNA expression
vector into cells
may involve particle bombardment. This method depends on the ability to
accelerate DNA coated
microprojectiles to a high velocity allowing them to pierce cell membranes and
enter cells without killing
them (Klein et al., 1987). Several devices for accelerating small particles
have been developed. One such
1 S device relies on a high voltage discharge to generate an electrical
current, which in turn provides the
motive force (Yang et al., 1990). The microprojectiles used have consisted of
biologically inert
substances such as tungsten or gold beads.
Selected organs including the liver, skin, and muscle tissue of rats and mice
have been bombarded
in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical
exposure of the tissue or cells,
to eliminate any intervening tissue between the gun and the target organ. DNA
encoding a Crrb2 or Crkl
construct may be delivered via this method.
Alternatively, the degree of cellular efflux in a cell may be influenced by
administering a
compound identified via one of the assays described above, that increases or
decreases the rate of
cellular efflux or development of MDR phenotype.
2S D. FORMULATION AND ADMINISTRATION OF THE DISCLOSED COMPOSITIONS
The formulations described herein may be prepared in water suitably mixed with
a surfactant,
such as hydroxypropylcellulose or polyoxyethylenesorbitans. In many cases, it
will be preferable to
include isotonic agents, for example, sugars or sodium chloride as described
above. Prolonged
absorption of the injectable compositions can be brought about by the use in
the compositions of
agents delaying absorption, for example, aluminum monostearate or gelatin.
Other agents that may be
employed include, but are not limited to lecithin, urea, ethylene oxide,
propylene oxide,
hydroxypropylcellulose, methylcellulose, or polyethylene glycol.
Aqueous compositions (inocula) as described herein may include an effective
amount of a
desired pharmacologically active agent dissolved or dispersed in a
pharmaceutically acceptable
3S aqueous medium. Such compositions are also referred to as inocula. The use
ofpharmaceutically
CA 02384104 2002-03-06
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acceptable carrier media and agents for pharmaceutically active substances is
well known in the art.
Except insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active ingredients
also can be incorporated into
the compositions as described above.
A semaphorin used in the present invention may be formulated into a
composition in a neutral
or salt form. Pharmaceutically acceptable salts include the acid addition
salts (formed with the free
amino groups of the protein) and those that are formed with inorganic acids
such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the
like. Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for
example, sodium, potassium, ammonium, calcium, or fernc hydroxides, and such
organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like.
Such compositions of the present invention can be, alternatively, complexed
with polyethylene
glycol (PEG), metal ions, or incorporated into polymeric compounds such as
polyacetic acid,
polyglycolic acid, hydrogels, dextran, etc. or incorporated into liposomes,
microemulsions, micelles,
1 S unilamellar or multilamellar vesicles, erythrocyte ghosts or
sphereoblasts. Such compositions will
influence the physical state, solubility, stability, rate of in vivo release,
and rate of in vivo clearance,
and are thus chosen according to the intended application.
The therapeutic compositions of the present invention are advantageously
administered in the
form of injectable compositions either as liquid solutions or suspensions;
solid forms suitable for solution
in, or suspension in, liquid prior to injection may also be prepared.
Alternatively, the compositions of the
present invention may be administered as inhalants in an aerosolized form.
Depending upon the needs of
the formulator, administrator, or the subject of the treatment, the presently
disclosed compositions may
take virtually any form including liquid, suspension, aerosol, emulsion,
solution, oil, mixture, cream,
ointment, gel, suppository, semi-solid, aerosol, powder, lyophilized form that
may be reconstituted when
appropriate, tablet, capsule or any other form or state convenient for
administration to treat the
described disorders. A typical composition comprises a pharmaceutically
acceptable carrier.
The presently disclosed compositions and methods may utilize both oral and non-
oral
administration routes to influence the target cell or cells including, for
example, by injection via the
intradermal, subcutaneous, and intravenous routes; by transdermal delivery; by
inhalation or buccal
delivery, or by ingestion of tablets or capsules. For example, local or
regional delivery of compounds
to a cell or cells can be by injection into the tissue, injection into the
vasculature or lymphatics to
effect regional infusion, inhalation, or regional perfusion by use of an
extracorporeal circuit.
Administration in a targeted fashion is useful to, for example, more
effectively eliminate neoplastic
cells, while minimizing the adverse effects of chemotherapy on healthy cells.
For example, an
inhibitor of cellular efflux can be directly administered, according to the
methods disclosed herein, to
21
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neoplastic cells such as tumor cells, to prevent their development of MDR and
thereby promote their
susceptibility to chemotherapeutic or otherwise cytotoxic agents, while
simultaneously administering
to healthy cells a promoter of cellular-efflux to prevent their destruction by
cytotoxic agents.
The optimal daily dose of semaphorin, semaphorin receptor such as VESPR or
soluble
VESPR, or of an agonist or antagonist of one of these, alone or in
combination, useful for the purposes
of the present invention is determined by methods known in the art. For
example, dosages can be
determined based on the severity of the disease or condition being treated,
the condition of the subject
to whom treatment is being given, the desired degree of therapeutic response,
and any concomitant
therapies being administered to the subject. Ordinarily, however,
administration will be such that a
serum level of between about 100ng/ml to about 1 OOpg/ml of semaphorin,
semaphorin receptor, or
agonist or antagonist of either, is achieved. Preferred doses will achieve
blood serum levels of
between SOOng/ml and 1 ug/ml. The dose can be administered in a single or
multiple dosage regimen,
or may be by a method that allows for a continuous release of relatively small
amounts of the active
ingredient from a single dosage unit, such as by a transdermal patch or
ingested extended release
capsule, over the course of one or more days.
To determine when inhibition or retardation of the various target diseases or
conditions, or
when amelioration, regression or destruction of the targeted diseases or
conditions has been achieved,
any of the following can be considered: improvement in patient condition or
quality of life; increased
longevity of life; decreased pain; decreased severity of symptoms of the
targeted disease or condition;
retardation of abnormal tissue growth or metastases such as in the case of
suppression of development
of MDR in cells being targeted for cancer chemotherapeutic disease; an
increase in desired tissue
growth or viability in the case or promotion of drug resistance in healthy
tissue and cells; and the like.
Any of these endpoints as well as others may be considered to determine the
effectiveness of the
therapy, and may be measured or determined by patient self evaluation;
objective screening; or by
diagnostic testing such as by X-ray, CT or PET scanning or the like.
The compositions as described herein may be formulated so that they are
contained in a vial,
bottle, tube, syringe inhaler or other container for single or multiple
administrations. Such containers
may be made of glass or a polymer material such as polypropylene,
polyethylene, or
polyvinylchloride, for example. Preferred containers may include a seal, or
other closure system, such
as a rubber stopper that may be penetrated by a needle in order to withdraw a
single dose and then re-
seal upon removal of the needle. All such containers for injectable liquids,
lyophilized formulations,
reconstituted lyophilized formulations or reconstitutable powders for
injection known in the art or for
the administration of aerosolized compositions are contemplated for use in the
presently disclosed
compositions and methods.
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In alternative embodiments, the presently disclosed compositions are
administered in
conjunction, either simultaneously or sequentially, with additional active
agents such as an
immunosuppressant, cell sensitizer, or other chemotherapeutic agent including
a cancer
chemotherapeutic agent. Exemplary agents to be used in combination with the
presently disclosed
compositions include cyclosporin, tamoxifen, FK506, taxotere, doxorubicin, cis-
platin, I-phosphamide,
or methotrexate.
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REFERENCES
Altan, et al., J. Exp. Med. 187:1583
Altan, et al., PNAS 96:4432
Chen, et al., JBC 274:18364
Kim et al., Blood, 91:4106-4117 (1998)
Lelong, et al., Molecular Pharmacology 40:490 (1991).
Schindler, et al., Biochemistry 35:2811
Weisburg et al, J. Biol Chem, 274, 10877-88 (1999)
Yamada et.al., PNAS 94:14713-14718 (1997)
Zaman, G.J.R. et.al., PNAS USA 91:8822 (1994)
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CA 02384104 2002-03-06
WO 01/18044 PCT/LTS00/24560
INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4707 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..4707
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATG GAG GTC TCC CGG AGG AAG GCG CCG CCG CGC CCC CCG CGC CCC GCA 48
Met Glu Val Ser Arg Arg Lys Ala Pro Pro Arg Pro Pro Arg Pro Ala
1 5 10 15
GCG CCA CTG CCC CTG CTC GCC TAT CTG CTG GCA CTG GCG GCT CCC GGC 96
Ala Pro Leu Pro Leu Leu Ala Tyr Leu Leu Ala Leu Ala Ala Pro Gly
20 25 30
CGG GGC GCG GAC GAG CCC GTG TGG CGG TCG GAG CAA GCC ATC GGA GCC 144
Arg Gly Ala Asp Glu Pro Val Trp Arg Ser Glu Gln Ala Ile Gly Ala
35 40 45
ATC GCG GCG AGC CAG GAG GAC GGC GTG TTT GTG GCG AGC GGC AGC TGC 192
Ile Ala Ala Ser Gln Glu Asp Gly Val Phe Val Ala Ser Gly Ser Cys
50 55 60
CTG GAC CAG CTG GAC TAC AGC CTG GAG CAC AGC CTC TCG CGC CTG TAC 240
Leu Asp Gln Leu Asp Tyr Ser Leu Glu His Ser Leu Ser Arg Leu Tyr
65 70 75 80
CGG GAC CAA GCG GGC AAC TGC ACA GAG CCG GTC TCG CTG GCG CCC CCC 288
Arg Asp Gln Ala Gly Asn Cys Thr Glu Pro Val Ser Leu Ala Pro Pro
85 90 95
GCG CGG CCC CGG CCC GGG AGC AGC TTC AGC AAG CTG CTG CTG CCC TAC 336
Ala Arg Pro Arg Pro Gly Ser Ser Phe Ser Lys Leu Leu Leu Pro Tyr
100 105 110
CGC GAG GGG GCG GCC GGC CTC GGG GGG CTG CTG CTC ACC GGC TGG ACC 384
Arg Glu Gly Ala Ala Gly Leu Gly Gly Leu Leu Leu Thr Gly Trp Thr
115 120 125
TTC GAC CGG GGC GCC TGC GAG GTG CGG CCC CTG GGC AAC CTG AGC CGC 432
1
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Phe Asp Arg Gly Ala Cys Glu Val Arg Pro Leu Gly Asn Leu
Ser Arg
130 135 140
AAC TCC CTG CGC AAC GGC ACC GAG GTG GTG TCG TGC CAC CCG 480
CAG GGC
Asn Ser Leu Arg Asn Gly Thr Glu Val Val Ser Cys His Pro
Gln Gly
145 150 155
160
TCG ACG GCC GGC GTG GTG TAC CGC GCG GGC CGG AAC AAC CGC 528
TGG TAC
Ser Thr Ala Gly Val Val Tyr Arg Ala Gly Arg Asn Asn Arg
Trp Tyr
165 170 175
CTG GCG GTG GCC GCC ACC TAC GTG CTG CCT GAG CCG GAG ACG 576
GCG AGC
Leu Ala Val Ala Ala Thr Tyr Val Leu Pro Glu Pro Glu Thr
Ala Ser
180 185 190
CGC TGC AAC CCC GCG GCA TCC GAC CAC GAC ACG GCC ATC GCG 624
CTC AAG
Arg Cys Asn Pro Ala Ala Ser Asp His Asp Thr Ala Ile Ala
Leu Lys
195 200 205
GAC ACG GAG GGG CGC AGC CTG GCC ACG CAG GAG CTG GGG CGC 672
CTC AAG
Asp Thr Glu Gly Arg Ser Leu Ala Thr Gln Glu Leu Gly Arg
Leu Lys
210 215 220
CTG TGC GAG GGC GCG GGC AGC CTG CAC TTC GTG GAC GCC TTT 720
CTC TGG
Leu Cys Glu Gly Ala Gly Ser Leu His Phe Val Asp Ala Phe
Leu Trp
225 230 235
240
AAC GGC AGC ATC TAC TTC CCC TAC TAC CCC TAC AAC TAT ACG 768
AGC GGC
Asn Gly Ser Ile Tyr Phe Pro Tyr Tyr Pro Tyr Asn Tyr Thr
Ser Gly
245 250 255
GCT GCC ACC GGC TGG CCC AGC ATG GCG CGC ATC GCG CAG AGC 816
ACC GAG
Ala Ala Thr Gly Trp Pro Ser Met Ala Arg Ile Ala Gln Ser
Thr Glu
260 265 270
GTG CTG TTC CAG GGC CAG GCA TCC CTC GAC TGC GGC CAC GGC 864
CAC CCC
Val Leu Phe Gln Gly Gln Ala Ser Leu Asp Cys Gly His Gly
His Pro
275 280 285
GAC GGC CGC CGC CTG CTC CTC TCC TCC AGC CTA GTG GAG GCC 912
CTG GAC
Asp Gly Arg Arg Leu Leu Leu Ser Ser Ser Leu Val Glu Ala
Leu Asp
290 295 300
GTC TGG GCG GGA GTG TTC AGC GCG GCC GCT GGA GAG GGC CAG 960
GAG CGG
Val Trp Ala Gly Val Phe Ser Ala Ala Ala Gly Glu Gly Gln
Glu Arg
305 310 315
320
CGC TCC CCC ACC ACC ACG GCG CTC TGC CTC TTC AGA ATG AGT 1008
GAG ATC
Arg Ser Pro Thr Thr Thr Ala Leu Cys Leu Phe Arg Met Ser
Glu Ile
325 330 335
CAG GCG CGC GCC AAG AGG GTC AGC TGG GAC TTC AAG ACG GCC 1056
GAG AGC
Gln Ala Arg Ala Lys Arg Val Ser Trp Asp Phe Lys Thr Ala
Glu Ser
340 345 350
2
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CACTGC AAA GGG GATCAACCT GAA 1104
GAA AGA
GTC
CAA
CCA
ATC
GCA
TCA
HisCys LysGluGly AspGlnPro GluArgVal GlnPro Ile Ser
Ala
355 360 365
TCTACC TTGATCCAT TCCGACCTG ACATCCGTT TATGGC ACC GTA 1152
GTG
SerThr LeuIleHis SerAspLeu ThrSerVal TyrGly Thr Val
Val
370 375 380
ATGAAC AGGACTGTT TTATTCTTG GGGACTGGA GATGGC CAGTTACTT 1200
MetAsn ArgThrVal LeuPheLeu GlyThrGly AspGly GlnLeuLeu
385 390 395 400
AAGGTT ATTCTTGGT GAGAATTTG ACTTCAAAT TGTCCA GAGGTTATC 1248
LysVal IleLeuGly GluAsnLeu ThrSerAsn CysPro GluValIle
405 410 415
TATGAA ATTAAAGAA GAGACACCT GTTTTCTAC AAACTC GTTCCTGAT 1296
TyrGlu IleLysGlu GluThrPro ValPheTyr LysLeu ValProAsp
420 425 430
CCTGTG AAGAATATC TACATTTAT CTAACAGCT GGGAAA GAGGTGAGG 1344
ProVal LysAsnIle TyrIleTyr LeuThrAla GlyLys GluValArg
435 440 445
AGAATT CGTGTTGCA AACTGCAAT AAACATAAA TCCTGT TCGGAGTGT 1392
ArgIle ArgValAla AsnCysAsn LysHisLys SerCys SerGluCys
450 455 460
TTAACA GCCACAGAC CCTCACTGC GGTTGGTGC CATTCG CTACAAAGG 1440
LeuThr AlaThrAsp ProHisCys GlyTrpCys HisSer LeuGlnArg
465 470 475 480
TGCACT TTTCAAGGA GATTGTGTA CATTCAGAG AACTTA GAAAACTGG 1488
CysThr PheGlnGly AspCysVal HisSerGlu AsnLeu GluAsnTrp
485 490 495
CTGGAT ATTTCGTCT GGAGCAAAA AAGTGCCCT AAAATT CAGATAATT 1536
LeuAsp IleSerSer GlyAlaLys LysCysPro LysIle GlnIleIle
500 505 510
CGAAGC AGTAAAGAA AAGACTACA GTGACTATG GTGGGA AGCTTCTCT 1584
ArgSer SerLysGlu LysThrThr ValThrMet ValGly SerPheSer
515 520 525
CCAAGA CACTCAAAG TGCATGGTG AAGAATGTG GACTCT AGCAGGGAG 1632
ProArg HisSerLys CysMetVal LysAsnVal AspSer SerArgGlu
530 535 540
CTCTGC CAGAATAAA AGTCAGCCC AACCGGACC TGCACC TGTAGCATC 1680
LeuCys GlnAsnLys SerGlnPro AsnArgThr CysThr CysSerIle
545 550 555 560
CCAACC AGAGCAACC TACAAAGAT GTTTCAGTT GTCAAC GTGATGTTC 1728
ProThr ArgAlaThr TyrLysAsp ValSerVal ValAsn ValMetPhe
565 570 575
3
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TCC 1776
TTC
GGT
TCT
TGG
AAT
TTA
TCA
GAC
AGA
TTC
AAC
TTT
ACC
AAC
TGC
Ser Ser Asp Arg Asn Cys
Phe Trp Phe Asn
Gly Asn Phe Thr
Leu
Ser
580 585 590
TCA CCA TGC GTA GCG TGG 1824
TCA GCA GAA ACT
TTA GGC TGC
AAA
GAA
TGC
Ser Leu LysGlu Pro Cys Val Ala Trp
Ser Cys Ala Glu Thr
Gly Cys
595 600 605
TGT AGT GCAAGA TGT CAC CCC ACA GCT GAC CCT 1872
AAA AGG ATC TTC TGC
Cys LysSer AlaArg Cys His Pro Thr Ala Asp Pro
Arg Ile Phe Cys
610 615 620
TCT GATTAT GAGAGA CAG CAG TGT GTG GCT GAG AAG 1920
AAC GAA CCA GTC
Ser AspTyr GluArg Gln Gln Cys Val Ala Glu Lys
Asn Glu Pro Val
625 630 635 640
ACA TCAGGA GGAGGA CCC GAG AAC GGG AAC ACC AAC 1968
AGA AAG AAG AGA
Thr SerGly GlyGly Pro Glu Asn Gly Asn Thr Asn
Arg Lys Lys Arg
645 650 655
CAG GCTTTA CAGGTC TAC AAG TCC GAG CCA AAA GTA 2016
TTC ATT ATT CAG
Gln AlaLeu GlnVal Tyr Lys Ser Glu Pro Lys Val
Phe Ile Ile Gln
660 665 670
TCG ACATTA GGGAAA AAC ATA GTA GGA GCA TTT ACC 2064
AGC GTG ACG AAC
Ser ThrLeu GlyLys Asn Ile Val Gly Ala Phe Thr
Ser Val Thr Asn
675 680 685
CGG GCATCG AACATC ATG CTG AAA ACC AGT TGT GAT 2112
ACA ATC GGA ACC
Arg AlaSer AsnIle Met Leu Lys Thr Ser Cys Asp
Thr Ile Gly Thr
690 695 700
AAG GATGTG ATACAG AGC GTG CTA GAC ACC ATG AAA 2160
GTT CAT AAT CAC
Lys AspVal IleGln Ser Val Leu Asp Thr Met Lys
Val His Asn His
705 710 715 720
TTC TCTCTT CCATCA CGG GAA ATG GAT GTG ATC CAG 2208
AGC AAA AAG TGT
Phe SerLeu ProSer Arg Glu Met Asp Val Ile Gln
Ser Lys Lys Cys
725 730 735
TTT GATGGT GGGAAC TCT GTG GGA TTA TCC ATT GCT 2256
TGC TCT TCC TAC
Phe AspGly GlyAsn Ser Val Gly Leu Ser Ile Ala
Cys Ser Ser Tyr
740 745 75p
CTG CCACAT TGTTCC ATA CCT GCT ACC TGG 2304
CTT TTT ACC ATC AGT
GGT
Leu ProHis CysSer Ile Pro Ala Thr Trp Ser Gly
Leu Phe Thr Ile
755 760 765
GGT CAA ACC ATG GAT GTA 2352
AAT ATG GGC ATT GAC
ATA AGA AAC
AAT
TTT
Gly GlnAsn IleThr Met Asp Val
Met Gly Ile Asp
Arg Asn
Asn
Phe
770 775 780
TTA ATC CAT 2400
ATT GAA
TCA TTA
AAA
GGA
AAC
ATA
AAT
GTC
TCT
GAA
TAT
Leu IleIle Leu
Ser Lys
His Gly
Glu Asn
Ile
Asn
Val
Ser
Glu
Tyr
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785 790 795 800
TGT GTG GCG ACT TAC TGC GGG TTT TTA GCC CCC AGT TTA AAG AGT TCA 2448
Cys Val Ala Thr Tyr Cys Gly Phe Leu Ala Pro Ser Leu Lys Ser Ser
805 810 815
AAA GTG CGC ACG AAT GTC ACT GTG AAG CTG AGA GTA CAA GAC ACC TAC 2496
Lys Val Arg Thr Asn Val Thr Val Lys Leu Arg Val Gln Asp Thr Tyr
820 825 830
TTG GAT TGT GGA ACC CTG CAG TAT CGG GAG GAC CCC AGA TTC ACG GGG 2544
Leu Asp Cys Gly Thr Leu Gln Tyr Arg Glu Asp Pro Arg Phe Thr Gly
835 840 845
TAT CGG GTG GAA TCC GAG GTG GAC ACA GAA CTG GAA GTG AAA ATT CAA 2592
Tyr Arg Val Glu Ser Glu Val Asp Thr Glu Leu Glu Val Lys Ile Gln
850 855 860
AAA GAA AAT GAC AAC TTC AAT ATT TCC AAA AAA GAC ATT GAA ATT ACT 2640
Lys Glu Asn Asp Asn Phe Asn Ile Ser Lys Lys Asp Ile Glu Ile Thr
865 870 875 880
CTC TTC CAT GGG GAA AAT GGG CAA TTA AAT TGC AGT TTT GAA AAT ATT 2688
Leu Phe His Gly Glu Asn Gly Gln Leu Asn Cys Ser Phe Glu Asn Ile
885 890 895
ACT AGA AAT CAA GAT CTT ACC ACC ATC CTT TGC AAA ATT AAA GGC ATC 2736
Thr Arg Asn Gln Asp Leu Thr Thr Ile Leu Cys Lys Ile Lys Gly Ile
900 905 910
AAG ACT GCA AGC ACC ATT GCC AAC TCT TCT AAG AAA GTT CGG GTC AAG 2784
Lys Thr Ala Ser Thr Ile Ala Asn Ser Ser Lys Lys Val Arg Val Lys
915 920 925
CTG GGA AAC CTG GAG CTC TAC GTC GAG CAG GAG TCA GTT CCT TCC ACA 2832
Leu Gly Asn Leu Glu Leu Tyr Val Glu Gln Glu Ser Val Pro Ser Thr
930 935 940
TGGTATTTTCTG ATTGTG CTCCCT GTCTTGCTAGTG ATTGTCATT TTT 2880
TrpTyrPheLeu IleVal LeuPro ValLeuLeuVal IleValIle Phe
945 950 955 960
GCGGCCGTGGGG GTGACC AGGCAC AAATCGAAGGAG CTGAGTCGC AAA 2928
AlaAlaValGly ValThr ArgHis LysSerLysGlu LeuSerArg Lys
965 970 975
CAGAGTCAACAA CTAGAA TTGCTG GAAAGCGAGCTC CGGAAAGAG ATA 2976
GlnSerGlnGln LeuGlu LeuLeu GluSerGluLeu ArgLysGlu Ile
980 985 990
CGTGACGGCTTT GCTGAG CTGCAG ATGGATAAATTG GATGTGGTT GAT 3024
ArgAspGlyPhe AlaGlu LeuGln MetAspLysLeu AspValVal Asp
995 1000 1005
AGT TTT GGA ACT GTT CCC TTC CTT GAC TAC AAA CAT TTT GCT CTG AGA 3072
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Ser Phe Gly Thr Val Pro Phe Leu Asp Tyr Lys His Phe Ala Leu Arg
1010 1015 1020
ACTTTC TTC CCTGAG GGT GGCTTC ACCCACATCTTC ACTGAA GAT 3120
TCA
ThrPhe Phe ProGlu Gly GlyPhe ThrHisIlePhe ThrGlu Asp
Ser
1025 1030 1035 1040
ATGCAT AAC AGAGAC AAC GACAAG AATGAAAGTCTC ACAGCT TTG 3168
GCC
MetHis Asn ArgAsp Asn AspLys AsnGluSerLeu ThrAla Leu
Ala
1045 1050 1055
GATGCC CTA ATCTGT AAA AGCTTT CTTGTTACTGTC ATCCAC ACC 3216
AAT
AspAla Leu IleCys Lys SerPhe LeuValThrVal IleHis Thr
Asn
1060 1065 1070
CTTGAA AAG CAGAAG TTT TCTGTG AAGGACAGGTGT CTGTTT GCC 3264
AAC
LeuGlu Lys GlnLys Phe SerVal LysAspArgCys LeuPhe Ala
Asn
1075 1080 1085
TCC TTC CTA ACC ATT GCA CTG CAA ACC AAG CTG GTC TAC CTG ACC AGC 3312
Ser Phe Leu Thr Ile Ala Leu Gln Thr Lys Leu Val Tyr Leu Thr Ser
1090 1095 1100
ATC CTA GAG GTG CTG ACC AGG GAC TTG ATG GAA CAG TGT AGT AAC ATG 3360
Ile Leu Glu Val Leu Thr Arg Asp Leu Met Glu Gln Cys Ser Asn Met
1105 1110 1115 1120
CAG CCG AAA CTC ATG CTG AGA CGC ACG GAG TCC GTC GTC GAA AAA CTC 3408
Gln Pro Lys Leu Met Leu Arg Arg Thr Glu Ser Val Val Glu Lys Leu
1125 1130 1135
CTC ACA AAC TGG ATG TCC GTC TGC CTT TCT GGA TTT CTC CGG GAG ACT 3456
Leu Thr Asn Trp Met Ser Val Cys Leu Ser Gly Phe Leu Arg Glu Thr
1140 1145 1150
GTC GGA GAG CCC TTC TAT TTG CTG GTG ACG ACT CTG AAC CAG AAA ATT 3504
Val Gly Glu Pro Phe Tyr Leu Leu Val Thr Thr Leu Asn Gln Lys Ile
1155 1160 1165
AAC AAG GGT CCC GTG GAT GTA ATC ACT TGC AAA GCC CTG TAC ACA CTT 3552
Asn Lys Gly Pro Val Asp Val Ile Thr Cys Lys Ala Leu Tyr Thr Leu
1170 1175 1180
AAT GAA GAC TGG CTG TTG TGG CAG GTT CCG GAA TTC AGT ACT GTG GCA 3600
Asn Glu Asp Trp Leu Leu Trp Gln Val Pro Glu Phe Ser Thr Val Ala
1185 1190 1195 1200
TTA AAC GTC GTC TTT GAA AAA ATC CCG GAA AAC GAG AGT GCA GAT GTC 3648
Leu Asn Val Val Phe Glu Lys Ile Pro Glu Asn Glu Ser Ala Asp Val
1205 1210 1215
TGT CGG AAT ATT TCA GTC AAT GTT CTC GAC TGT GAC ACC ATT GGC CAA 3696
Cys Arg Asn Ile Ser Val Asn Val Leu Asp Cys Asp Thr Ile Gly Gln
1220 1225 1230
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GCC AAA GAA AAG ATT TTC CAA GCA TTC TTA AGC AAA AAT GGC TCT CCT 3744
Ala Lys Glu Lys Ile Phe Gln Ala Phe Leu Ser Lys Asn Gly Ser Pro
1235 1240 1245
TAT GGA CTT CAG CTT AAT GAA ATT GGT CTT GAG CTT CAA ATG GGC ACA 3792
Tyr Gly Leu Gln Leu Asn Glu Ile Gly Leu Glu Leu Gln Met Gly Thr
1250 1255 1260
CGA CAG AAA GAA CTT CTG GAC ATC GAC AGT TCC TCC GTG ATT CTT GAA 3840
Arg Gln Lys Glu Leu Leu Asp Ile Asp Ser Ser Ser Val Ile Leu Glu
1265 1270 1275 1280
GAT GGA ATC ACC AAG CTA AAC ACC ATT GGC CAC TAT GAG ATA TCA AAT 3888
Asp Gly Ile Thr Lys Leu Asn Thr Ile Gly His Tyr Glu Ile Ser Asn
1285 1290 1295
GGA TCC ACT ATA AAA GTC TTT AAG AAG ATA GCA AAT TTT ACT TCA GAT 3936
Gly Ser Thr Ile Lys Val Phe Lys Lys Ile Ala Asn Phe Thr Ser Asp
1300 1305 1310
GTG GAG TAC TCG GAT GAC CAC TGC CAT TTG ATT TTA CCA GAT TCG GAA 3984
Val Glu Tyr Ser Asp Asp His Cys His Leu Ile Leu Pro Asp Ser Glu
1315 1320 1325
GCA TTC CAA GAT GTG CAA GGA AAG AGA CAT CGA GGG AAG CAC AAG TTC 4032
Ala Phe Gln Asp Val Gln Gly Lys Arg His Arg Gly Lys His Lys Phe
1330 1335 1340
AAA GTA AAA GAA ATG TAT CTG ACA AAG CTG CTG TCG ACC AAG GTG GCA 4080
Lys Val Lys Glu Met Tyr Leu Thr Lys Leu Leu Ser Thr Lys Val Ala
1345 1350 1355 1360
ATT CAT TCT GTG CTT GAA AAA CTT TTT AGA AGC ATT TGG AGT TTA CCC 4128
Ile His Ser Val Leu Glu Lys Leu Phe Arg Ser Ile Trp Ser Leu Pro
1365 1370 1375
AAC AGC AGA GCT CCA TTT GCT ATA AAA TAC TTT TTT GAC TTT TTG GAC 4176
Asn Ser Arg Ala Pro Phe Ala Ile Lys Tyr Phe Phe Asp Phe Leu Asp
1380 1385 1390
GCC CAG GCT GAA AAC AAA AAA ATC ACA GAT CCT GAC GTC GTA CAT ATT 4224
Ala Gln Ala Glu Asn Lys Lys Ile Thr Asp Pro Asp Val Val His Ile
1395 1400 1405
TGG AAA ACA AAC AGC CTT CCT CTT CGC TTC TGG GTA AAC ATC CTG AAG 4272
Trp Lys Thr Asn Ser Leu Pro Leu Arg Phe Trp Val Asn Ile Leu Lys
1410 1415 1420
AAC CCT CAG TTT GTC TTT GAC ATT AAG AAG ACA CCA CAT ATA GAC GGC 4320
Asn Pro Gln Phe Val Phe Asp Ile Lys Lys Thr Pro His Ile Asp Gly
1425 1430 1435 1440
TGT TTG TCA GTG ATT GCC CAG GCA TTC ATG GAT GCA TTT TCT CTC ACA 4368
Cys Leu Ser Val Ile Ala Gln Ala Phe Met Asp Ala Phe Ser Leu Thr
1445 1450 1455
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GAGCAGCAA CTAGGG AAGGAAGCA CCA AAT AAGCTTCTC TATGCC 4416
ACT
GluGlnGln LeuGly LysGluAla ProThrAsn LysLeuLeu TyrAla
1460 1465 1470
AAGGATATC CCAACC TACAAAGAA GAAGTAAAA TCTTATTAC AAAGCA 4464
LysAspIle ProThr TyrLysGlu GluValLys SerTyrTyr LysAla
1475 1480 1485
ATCAGGGAT TTGCCT CCATTGTCA TCCTCAGAA ATGGAAGAA TTTTTA 4512
IleArgAsp LeuPro ProLeuSer SerSerGlu MetGluGlu PheLeu
1490 1495 1500
ACTCAGGAA TCTAAG AAACATGAA AATGAATTT AATGAAGAA GTGGCC 4560
ThrGlnGlu SerLys LysHisGlu AsnGluPhe AsnGluGlu ValAla
1505 1510 1515 1520
TTGACAGAA ATTTAC AAATACATC GTAAAATAT TTTGATGAG ATTCTA 4608
LeuThrGlu IleTyr LysTyrIle ValLysTyr PheAspGlu IleLeu
1525 1530 1535
AATAAACTA GAAAGA GAACGAGGG CTGGAAGAA GCTCAGAAA CAACTC 4656
AsnLysLeu GluArg GluArgGly LeuGluGlu AlaGlnLys GlnLeu
1540 1545 1550
TTGCATGTA AAAGTC TTATTTGAT GAAAAGAAG AAATGCAAG TGGATG 4704
LeuHisVal LysVal LeuPheAsp GluLysLys LysCysLys TrpMet
1555 1560 1565
TAA
4707
INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1569 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Glu Val Ser Arg Arg Lys Ala Pro Pro Arg Pro Pro Arg Pro Ala
1 5 10 15
Ala Pro Leu Pro Leu Leu Ala Tyr Leu Leu Ala Leu Ala Ala Pro Gly
20 25 30
Arg Gly Ala Asp Glu Pro Val Trp Arg Ser Glu Gln Ala Ile Gly Ala
35 40 45
Ile Ala Ala Ser Gln Glu Asp Gly Val Phe Val Ala Ser Gly Ser Cys
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50 55 60
Leu Asp Gln Leu Asp Tyr Ser Leu Glu His Ser Leu Ser Arg Leu Tyr
65 70 75 gp
Arg Asp Gln Ala Gly Asn Cys Thr Glu Pro Val Ser Leu Ala Pro Pro
85 90 95
Ala Arg Pro Arg Pro Gly Ser Ser Phe Ser Lys Leu Leu Leu Pro Tyr
100 105 110
Arg Glu Gly Ala Ala Gly Leu Gly Gly Leu Leu Leu Thr Gly Trp Thr
115 120 125
Phe Asp Arg Gly Ala Cys Glu Val Arg Pro Leu Gly Asn Leu Ser Arg
130 135 140
Asn Ser Leu Arg Asn Gly Thr Glu Val Val Ser Cys His Pro Gln Gly
145 150 155 160
Ser Thr Ala Gly Val Val Tyr Arg Ala Gly Arg Asn Asn Arg Trp Tyr
165 170 175
Leu Ala Val Ala Ala Thr Tyr Val Leu Pro Glu Pro Glu Thr Ala Ser
180 185 190
Arg Cys Asn Pro Ala Ala Ser Asp His Asp Thr Ala Ile Ala Leu Lys
195 200 205
Asp Thr Glu Gly Arg Ser Leu Ala Thr Gln Glu Leu Gly Arg Leu Lys
210 215 220
Leu Cys Glu Gly Ala Gly Ser Leu His Phe Val Asp Ala Phe Leu Trp
225 230 235 240
Asn Gly Ser Ile Tyr Phe Pro Tyr Tyr Pro Tyr Asn Tyr Thr Ser Gly
245 250 255
Ala Ala Thr Gly Trp Pro Ser Met Ala Arg Ile Ala Gln Ser Thr Glu
260 265 270
Val Leu Phe Gln Gly Gln Ala Ser Leu Asp Cys Gly His Gly His Pro
275 280 285
Asp Gly Arg Arg Leu Leu Leu Ser Ser Ser Leu Val Glu Ala Leu Asp
290 295 300
Val Trp Ala Gly Val Phe Ser Ala Ala Ala Gly Glu Gly Gln Glu Arg
305 310 315 320
Arg Ser Pro Thr Thr Thr Ala Leu Cys Leu Phe Arg Met Ser Glu Ile
325 330 335
Gln Ala Arg Ala Lys Arg Val Ser Trp Asp Phe Lys Thr Ala Glu Ser
340 345 350
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His Cys Lys Glu Gly Asp Gln Pro Glu Arg Val Gln Pro Ile Ala Ser
355 360 365
Ser Thr Leu Ile His Ser Asp Leu Thr Ser Val Tyr Gly Thr Val Val
370 375 380
Met Asn Arg Thr Val Leu Phe Leu Gly Thr Gly Asp Gly Gln Leu Leu
385 390 395 400
Lys Val Ile Leu Gly Glu Asn Leu Thr Ser Asn Cys Pro Glu Val Ile
405 410 415
Tyr Glu Ile Lys Glu Glu Thr Pro Val Phe Tyr Lys Leu Val Pro Asp
420 425 430
Pro Val Lys Asn Ile Tyr Ile Tyr Leu Thr Ala Gly Lys Glu Val Arg
435 440 445
Arg Ile Arg Val Ala Asn Cys Asn Lys His Lys Ser Cys Ser Glu Cys
450 455 460
Leu Thr Ala Thr Asp Pro His Cys Gly Trp Cys His Ser Leu Gln Arg
465 470 475 480
Cys Thr Phe Gln Gly Asp Cys Val His Ser Glu Asn Leu Glu Asn Trp
485 490 495
Leu Asp Ile Ser Ser Gly Ala Lys Lys Cys Pro Lys Ile Gln Ile Ile
500 505 510
Arg Ser Ser Lys Glu Lys Thr Thr Val Thr Met Val Gly Ser Phe Ser
515 520 525
Pro Arg His Ser Lys Cys Met Val Lys Asn Val Asp Ser Ser Arg Glu
530 535 540
Leu Cys Gln Asn Lys Ser Gln Pro Asn Arg Thr Cys Thr Cys Ser Ile
545 550 555 560
Pro Thr Arg Ala Thr Tyr Lys Asp Val Ser Val Val Asn Val Met Phe
565 570 575
Ser Phe Gly Ser Trp Asn Leu Ser Asp Arg Phe Asn Phe Thr Asn Cys
580 585 590
Ser Ser Leu Lys Glu Cys Pro Ala Cys Val Glu Thr Gly Cys Ala Trp
595 600 605
Cys Lys Ser Ala Arg Arg Cys Ile His Pro Phe Thr Ala Cys Asp Pro
610 615 620
Ser Asp Tyr Glu Arg Asn Gln Glu Gln Cys Pro Val Ala Val Glu Lys
625 630 635 640
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Thr Ser Gly Gly Gly Arg Pro Lys Glu Asn Lys Gly Asn Arg Thr Asn
645 650 655
Gln Ala Leu Gln Val Phe Tyr Ile Lys Ser Ile Glu Pro Gln Lys Val
660 665 670
Ser Thr Leu Gly Lys Ser Asn Val Ile Val Thr Gly Ala Asn Phe Thr
675 680 685
Arg Ala Ser Asn Ile Thr Met Ile Leu Lys Gly Thr Ser Thr Cys Asp
690 695 700
Lys Asp Val Ile Gln Val Ser His Val Leu Asn Asp Thr His Met Lys
705 710 715 720
Phe Ser Leu Pro Ser Ser Arg Lys Glu Met Lys Asp Val Cys Ile Gln
725 730 735
Phe Asp Gly Gly Asn Cys Ser Ser Val Gly Ser Leu Ser Tyr Ile Ala
740 745 750
Leu Pro His Cys Ser Leu Ile Phe Pro Ala Thr Thr Trp Ile Ser Gly
755 760 765
Gly Gln Asn Ile Thr Met Met Gly Arg Asn Phe Asp Val Ile Asp Asn
770 775 780
Leu Ile Ile Ser His Glu Leu Lys Gly Asn Ile Asn Val Ser Glu Tyr
785 790 795 800
Cys Val Ala Thr Tyr Cys Gly Phe Leu Ala Pro Ser Leu Lys Ser Ser
805 810 815
Lys Val Arg Thr Asn Val Thr Val Lys Leu Arg Val Gln Asp Thr Tyr
820 825 830
Leu Asp Cys Gly Thr Leu Gln Tyr Arg Glu Asp Pro Arg Phe Thr Gly
835 840 845
Tyr Arg Val Glu Ser Glu Val Asp Thr Glu Leu Glu Val Lys Ile Gln
850 855 860
Lys Glu Asn Asp Asn Phe Asn Ile Ser Lys Lys Asp Ile Glu Ile Thr
865 870 875 880
Leu Phe His Gly Glu Asn Gly Gln Leu Asn Cys Ser Phe Glu Asn Ile
885 890 895
Thr Arg Asn Gln Asp Leu Thr Thr Ile Leu Cys Lys Ile Lys Gly Ile
900 905 910
Lys Thr Ala Ser Thr Ile Ala Asn Ser Ser Lys Lys Val Arg Val Lys
915 920 925
Leu Gly Asn Leu Glu Leu Tyr Val Glu Gln Glu Ser Val Pro Ser Thr
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930 935 940
Trp Tyr Phe Leu Ile Val Leu Pro Val Leu Leu Val Ile Val Ile Phe
945 950 955
960
Ala Ala Val Gly Val Thr Arg His Lys Ser Lys Glu Leu Ser Arg Lys
965 970 975
Gln Ser Gln Gln Leu Glu Leu Leu Glu Ser Glu Leu Arg Lys Glu Ile
980 985 990
Arg Asp Gly Phe Ala Glu Leu Gln Met Asp Lys Leu Asp Val Val Asp
995 1000
1005
Ser Phe Gly Thr Val Pro Phe Leu Asp Tyr Lys His Phe Ala Leu Arg
1010 1015 1020
Thr Phe Phe Pro Glu Ser Gly Gly Phe Thr His Ile Phe Thr Glu Asp
1025 1030 1035
1040
Met His Asn Arg Asp Ala Asn Asp Lys Asn Glu Ser Leu Thr Ala Leu
1045 1050 1055
Asp Ala Leu Ile Cys Asn Lys Ser Phe Leu Val Thr Val Ile His Thr
1060 1065
1070
Leu Glu Lys Gln Lys Asn Phe Ser Val Lys Asp Arg Cys Leu Phe Ala
1075 1080
1085
Ser Phe Leu Thr Ile Ala Leu Gln Thr Lys Leu Val Tyr Leu Thr Ser
1090 1095
1100
Ile Leu Glu Val Leu Thr Arg Asp Leu Met Glu Gln Cys Ser Asn Met
1105 1110 1115
1120
Gln Pro Lys Leu Met Leu Arg Arg Thr Glu Ser Val Val Glu Lys Leu
1125 1130
1135
Leu Thr Asn Trp Met Ser Val Cys Leu Ser Gly Phe Leu Arg Glu Thr
1140 1145 1150
Val Gly Glu Pro Phe Tyr Leu Leu Val Thr Thr Leu Asn Gln Lys Ile
1155 1160 1165
Asn Lys Gly Pro Val Asp Val Ile Thr Cys Lys Ala Leu Tyr Thr Leu
1170 1175 1180
Asn Glu Asp Trp Leu Leu Trp Gln Val Pro Glu Phe Ser Thr Val Ala
1185 1190 1195
1200
Leu Asn Val Val Phe Glu Lys Ile Pro Glu Asn Glu Ser Ala Asp Val
1205 1210 1215
Cys Arg Asn Ile Ser Val Asn Val Leu Asp Cys Asp Thr Ile Gly Gln
1220 1225 1230
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Ala Lys Glu Lys Ile Phe Gln Ala Phe Leu Ser Lys Asn Gly Ser Pro
1235 1240
1245
Tyr Gly Leu Gln Leu Asn Glu Ile Gly Leu Glu Leu Gln Met Gly Thr
1250 1255 1260
Arg Gln Lys Glu Leu Leu Asp Ile Asp Ser Ser Ser Val Ile Leu Glu
1265 1270 1275
1280
Asp Gly Ile Thr Lys Leu Asn Thr Ile Gly His Tyr Glu Ile Ser Asn
1285 1290 1295
Gly Ser Thr Ile Lys Val Phe Lys Lys Ile Ala Asn Phe Thr Ser Asp
1300 1305 1310
Val Glu Tyr Ser Asp Asp His Cys His Leu Ile Leu Pro Asp Ser Glu
1315 1320 1325
Ala Phe Gln Asp Val Gln Gly Lys Arg His Arg Gly Lys His Lys Phe
1330 1335
1340
Lys Val Lys Glu Met Tyr Leu Thr Lys Leu Leu Ser Thr Lys Val Ala
1345 1350 1355
1360
Ile His Ser Val Leu Glu Lys Leu Phe Arg Ser Ile Trp Ser Leu Pro
1365 1370 1375
Asn Ser Arg Ala Pro Phe Ala Ile Lys Tyr Phe Phe Asp Phe Leu Asp
1380 1385 1390
Ala Gln Ala Glu Asn Lys Lys Ile Thr Asp Pro Asp Val Val His Ile
1395 1400 1405
Trp Lys Thr Asn Ser Leu Pro Leu Arg Phe Trp Val Asn Ile Leu Lys
1410 1415 1420
Asn Pro Gln Phe Val Phe Asp Ile Lys Lys Thr Pro His Ile Asp Gly
1425 1430 1435 1440
Cys Leu Ser Val Ile Ala Gln Ala Phe Met Asp Ala Phe Ser Leu Thr
1445 1450 1455
Glu Gln Gln Leu Gly Lys Glu Ala Pro Thr Asn Lys Leu Leu Tyr Ala
1460 1465 1470
Lys Asp Ile Pro Thr Tyr Lys Glu Glu Val Lys Ser Tyr Tyr Lys Ala
1475 1480 1485
Ile Arg Asp Leu Pro Pro Leu Ser Ser Ser Glu Met Glu Glu Phe Leu
1490 1495 1500
Thr Gln Glu Ser Lys Lys His Glu Asn Glu Phe Asn Glu Glu Val Ala
1505 1510 1515
1520
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Leu Thr Glu Ile Tyr Lys Tyr Ile Val Lys Tyr Phe Asp Glu Ile Leu
1525 1530
1535
Asn Lys Leu Glu Arg Glu Arg Gly Leu Glu Glu Ala Gln Lys Gln Leu
1540 1545 1550
Leu His Val Lys Val Leu Phe Asp Glu Lys Lys Lys Cys Lys Trp Met
1555 1560 1565
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